This work was supported by a BBSRC project grant to DMcK, DMcC and RGN (BB/K002775/1) and a PhD studentship [to JP, BB/J013854/1]. We gratefully acknowledge the fantastic advice on technical equipment and field techniques that we received throughout the project from Nozrat Mirzai (Bioelectronics Unit University of Glasgow), Ross McLeod and Matthew Clavey (ThermalVision Research) as well as SCENE for all their logistic support of the field work. We would also like to thank Debbie Kelton at Veracity for generously providing equipment used to power the thermal imaging camera in the field.

Ethics Statement:
The work involved a routine trapping method of free-living small passerines approved by the British Trust for Ornithology and the capture protocol eliciting a mild stress in the bird was carried out under UK Home Office licenses.
1. Set-up for Filming
<ol>
	<li>Create a filming set-up where birds are encouraged to position themselves in front of the camera (Figure 1). The bird enters the set-up through a hole in one end wall and has access to food close to the opposite end wall.</li>
	<li>In order to habituate the birds to the set-up, provide a suitable food (e.g., peanut kernels) at the feeder for several weeks prior to recording. Apart from provision of the food, leave the set-up undisturbed. During this period, place a dummy tripod in front of the feeder to allow the birds to also habituate to the camera.</li>
	<li>On the day of recording, reduce the availability of peanut kernels in the set-up by removing all remaining food from the feeder and replacing it with food contained in a transparent container with only a small hole at the center on the top through which birds can access the food. Position this food container in the center of the cage at the end opposite to the entrance hole.</li>
	<li>Place a small square of black insulation tape of known emissivity to that of natural integument in one of the corners of the mesh of the box so it appears in all images. Attach the black insulation tape to a thermocouple connected with a temperature logger that records the temperature of the black insulation tape with a resolution of 0.1 &#176;C every 1 sec.</li>
	<li>Place the thermal imaging camera 50 cm from the center of the box trap so that all of the box fits within the camera&#8217;s field of view, and birds feeding from the clear container will be positioned in the camera&#8217;s zone of focus. Connect the camera to a laptop, set the camera to record images at on average of 7.5 frames per second (with a time stamp), and send images to the laptop&#8217;s hard disk to be saved there.</li>
	<li>Attach a fishing line to the rotating door of the set-up and roll it out to a position where the experimenter is hidden from view of the birds, but the set-up can still be observed&#160;&#8212; about 20 m from the set-up.</li>
</ol>
2. Filming the Bird&#8217;s Response to a Mild Acute Stress
<ol>
	<li>Once a bird enters the feeding box, leave the bird undisturbed in the box for ca. 5 sec.</li>
	<li>Pull the fishing line after the bird had spent ca. 5 sec in the box to close the feeding box. Take care that the bird is still at the far end of the box in order to minimize the risk of injury to the bird.</li>
	<li>Immediately approach the feeding box and stand motionless behind the camera for approximately 3 min. Then retrieve the bird from the box and let it go.</li>
</ol>
3. Extraction of Eye-region Temperature from Thermal Images
<ol>
	<li>Extract maximum temperature from each frame. Note: The maximum temperature was virtually always recorded from within the region around the eye, bounded by the exposed skin of the periophthalmic ring (Figure 2), and is hereafter referred to as eye-region temperature.
	<ol>
		<li>In the Thermal Imaging Analysis Software right click on image, &#8216;add&#8217; a new plot for the image maximum. Then, right click on the plot that exports the data (eye-region temperature and the time the frame was taken) to a CSV file.</li>
	</ol>
	</li>
	<li>Delete from the CSV file all lines from frames where the eye-region of the bird was not visible.</li>
	<li>Plot maximum temperature against time in R 27, identify manually points where temperature spikes &#62; +0.2 &#186;C between two successive readings when the nictitating membrane was pulled over the eye (blinking) and points with low values outside the range of body temperatures for birds 28 (i.e., when the eye-region was visible but blurred through motion for multiple frames) and then remove the relevant lines from the CSV data file produced during step 3.1.</li>
	<li>Measure ambient temperature.
	<ol>
		<li>Download the data (time and logger temperature) from the temperature logger onto a computer and export into a spreadsheet.</li>
		<li>To get the ambient temperature from the thermal image, in the Thermal Image Analysis Software draw a square over the black insulation tape that covered the temperature logger probe, then&#160;right click on the square and select &#8216;add new plot&#39; for the square&#39;s average temperature.</li>
		<li>Then, right click that plot, to export the data (time and IRT temperature) to a CSV file. Merge the two resulting temperature time series for temperature logger and thermal image matching for time into one spreadsheet.</li>
	</ol>
	</li>
	<li>Correct eye-region temperature against ambient temperature. Export the spreadsheet created in step 3.4 into the CSV file matching for time. To each retained eye-region temperature add the difference between the temperature logger and the thermal imaging-derived temperature values (logger temp &#8211; IRT temp) measured at the same time.</li>
	<li>Carry out automated filtering to remove less accurate low eye-region temperature values using the peak search algorithm (see Supplemental Code File) to extract the highest (and therefore most accurate) points in the data automatically. 
	Note: The peak search algorithm reorganizes the temperature data into a vector with a user-defined width (span), recommended span = 3, extracting the central value in rows where the numbers either side are lower, i.e., the peaks.</li>
	<li>Use linear interpolation to close the gaps left by sequences when no peak was extracted, and when the eye-region wasn&#39;t visible. Use the command na.approx (zoo package v1.7-11 in R 27) to give a single temperature value per second for each individual.</li>
	<li>In the CVS data file, add a value of 0.2 to each eye-region temperature to correct for the effect of taking the images through a mesh. 
	Note: Tests showed that when a black body, heated to approximate mean bird body temperature of ~41 &#186;C in its active phase 28, was imaged through the mesh window with the same mesh size as used in the filming set-up, temperatures recorded by the thermal imaging camera in areas not obscured by the wires were 0.2 &#186;C lower than values obtained without the intervening mesh (mean difference = -0.2 &#186;C &#177; 0.085 SD, n = 30, temperature range of black body = 41.6&#8211;42.5 &#186;C). 
	Note: This is the correction factor specific to the conditions in this study and is likely to vary with instrument make, and type and width of the mesh between the animal and the camera, and in each situation needs to be established for the specific study conditions.</li>
	<li>From the maximum eye-region temperatures recorded from before the box was closed select the highest value, which will constitute the baseline eye-region temperature of the undisturbed bird. Add this baseline eye-region temperature as a new column to the CVS file (baseline). Then subtract the baseline value from each retained maximum eye-region temperature value, generating a new column in the CVS data file. 
	Note: This new value now express the bird&#8217;s response to mild acute stress as deviation from its undisturbed baseline temperature.</li>
	<li>Plot the deviations in maximum eye-region temperature from the baseline eye-region temperature of all individuals from after the trap was closed using the command ggplot (package ggplot2 v1.0.0 in R 27). Generate bootstrapped 95% confidence intervals using the option mean_cl_boot (package ggplot2 v1.0.0 in R 27). For details see the Supplemental Code File.</li>
</ol>

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</ol>

The authors declare that they have no competing financial interests.

The aim of this paper is to describe the necessary methodology to measure changes in skin temperature of a free-living animal in response to an acute stressor. This study demonstrated that rapid changes in skin temperature associated with an acute stress response in wild birds can be captured non-invasively using IRT. This procedure involved five important stages (1) design of appropriate field set-up using a highly portable thermal imaging system, (2) measurement of baseline temperature, (3) application of a mild stressor involving capturing the bird in the set-up, (4) post hoc automated sampling of eye-region temperature and (5) calibration of extracted temperature data. The methodology described here was applied to the capture of wild blue tits. Representative baseline eye-region temperatures could be captured in as little as 5 sec. The estimated time required to record one bird in the field was about 1 hr, with a further hour required to process the 3 min thermal video sequence.
Collecting good data on an animal&#8217;s skin temperature requires high quality thermal images. Thermal imaging cameras only differ fundamentally from visible light cameras in terms of the wavelength of the electromagnetic radiation that they detect, and thus many of the concepts that apply in photography (e.g., field of view, depth of field) also apply in thermography. As commonly practiced by wildlife photographers, any location that an animal visits predictably, where natural (e.g., nest sites) or artificial (e.g., feeders), could be used for collecting thermal images. Attracting animals to the camera rather than attempting to follow them though their habitat has the advantage of allowing us to set up the camera in advance, to collect high-quality image with minimal disturbance as soon as the animal comes into view. However, with this approach, it is vital to examine or account for the effects of that specific context of measurement on stress or skin temperature. For example, here, birds were measured in a feeding context, and food was used to actively lure blue tits into the set-up. Research on chickens suggests that acute positive experiences, such as the anticipation and consumption of a food reward in an associative learning trial can also lower eye temperature 29. As such, &#8216;baseline levels&#8217; of individuals entering this particular set up may be influenced by the association of this act with reward. This possibility warrants further investigation, though if present, would be expected to enhance the drop from baseline levels already observed. Moreover, thermogenesis occurs with the digestion of food, though the specific effects of food consumed during baseline measurement are not expected to significantly elevate temperature within the 3 min trapping period 30.
This limitation considered, measuring all animals within the same context is also a great strength of this approach. The representative results showed that there was a large variation in eye-region temperature between individuals before the acute stressor was applied (baseline eye-region temperature). The variation in baseline eye temperature (coefficient of variation = 3.9%) could be partly due to measurement error or a true reflection of between-individual differences. The period the IRT camera recorded temperature of the black insulation tape over 5 sec when the tape&#39;s temperature did not change, the mean coefficient of variation was 0.26% (range 0.08% to 0.59%). Although measurement error is likely to be larger in a moving bird it nonetheless indicates that most of this variation in baseline eye-region temperature is indeed between individuals. The notion of relatively small measurement error on moving birds is further supported by the high repeatability of eye-region temperature over 20 sec during the baseline measurements (Figure 3). By controlling for context, we can ensure that all birds were engaging in the same activity. We can also minimize environmental contributions at the time of measurement to between-individual variation in skin temperature, For example, by ensuring that the locality is protected from confounding incident radiation that would, for thermal imaging, lead to an overestimation of the animal&#8217;s surface temperature. This can be done with working in the shade or by strategically placed shades, including most transparent plastics that block infra-red wavelength. The protocol presented here shows a simple set-up that could be easily applied to the situation of wintering blue tits. The blue tit was filmed while in a feeding box with a solid roof which avoided the exposure to direct sunlight. This set-up might also be applied to many other passerines that regularly come to artificial feeders at certain times of the year. As blue tits are comparatively small animals, they need to be filmed close-up to capture a good level of detail. As with photographic cameras, getting close to the subject &#8212; both in terms of actual distance, and magnification &#8212; dramatically reduces depth of field. To have the birds as large as possible in the field of view required filming them from the camera&#8217;s minimum focus distance, which in this case was 500 mm and reduced depth of field to 40 mm. Therefore, all features of the filming set-up need to be designed to encourage the animals to display their head within the relatively narrow zone of sharp focus of the particular camera used.
In response to a well-established acute mild stressor to an animal we expected rapid changes in the pattern of blood flow, from the periphery to the core via sympathetically mediated vasoconstriction that reduces skin temperature 17. The periorbital region was chosen as this provided the only region of the body that was uninsulated and is associated with a rich intermingled network of small blood vessels, the rete ophthalmicum that can affect the heat loss from the eye 31. The fast frame rate of the thermal videoing technique was able to show a drop in eye-region temperature of approximately 2 &#176;C in 10 sec followed by a subsequent rise in temperature to within 0.5 &#176;C of baseline within 3 min. Although peripheral cooling in response to mild stress has been recorded previously at around 1 min&#160;intervals 17,26, 32, the technique here showed that maximum drop in temperature may be extremely rapid. With lower time resolution the magnitude of this effect may be missed. This may be important when comparing differences in the stress response of different individuals. The re-warming of the bird after the rapid &#8216;fight or flight&#8217; response did not return to baseline level as the bird remained within the box indicating that the bird remained physiologically stressed. The birds were released from the experimental set-up after an arbitrarily chosen cut-off point of ca. 3 min&#160;to minimize the period of restraint of a free-living animal. However, in future, trials may need to be longer to record the complete temperature response until the animal&#8217;s eye-region temperature has returned to baseline level, if logistically possible.
This new protocol involving a series of images collected before and after the stressor was applied now allows detailed study of the dynamic of the response to acute stress, and comparisons of multiple time-points between individual animals. Repeating this trial within individuals over different seasons or environmental conditions may allow the effects of environment on baseline or post-acute stress skin temperature to be disentangled, as a possible avenue into chronic stress assessment as well. .
Skin temperature of a bird is dependent not only on metabolic heat production and blood flow but also on heat exchange from solar radiation, wind speed and wetting 25. This precludes wet and windy conditions from accurate thermographic recordings. The eye-region temperature recorded by a thermal imaging camera can at times either be underestimated (negative error) or overestimated (positive error). Substantial positive error would require energy input, but this was avoided by the bird in the feeding box being shielded from the sun. One other source of positive error included when the bird blinked. Occasionally the bird briefly pulls its nictitating membrane over the eye-region and as it is stored internally at a temperature closer to that of the body core, blinking gives an anomalously high temperature reading when it covers the eyeball. This is, however, easily detected as it leaves a very marked signature and the affected frame can be removed. The main reason for a negative error in our records was motion blur. Any movement too swift to be captured sharply by the frame rate of the camera confounds the data captured from the small warm eye-region of the image with that of the surrounding cool area (motion blur), resulting in an underestimation of eye-region temperature. Thus after removing positive errors this makes the maximum temperature measured from the eye-region the most accurate measurement and an automated filtering using the peaks function command removes the less likely lower values of maximum eye-region temperatures.
In addition, as infrared radiation is absorbed by water vapor, surface temperature recorded will be influenced by the relative humidity of the environment. This can be accounted for by entering air temperature, relative humidity and distance to object into the analytical Thermal Image Analysis Software. However, a more accurate and efficient approach (as undertaken here) is to include a reference body of known temperature and emissivity within the field of view. In our case this was a square of black insulation tape with an emissivity of 0.97, which is approximately equivalent to that of natural integument 33. This allows calibrating the eye-region temperature by using the difference between the actual and thermal imaging-derived temperature values measured for the insulation tape. The surface temperature of the blackbody can then be used to calibrate images continuously throughout the measurement period.
Thanks to the technological advances in the development in the thermal cameras they can now be deployed as small lightweight cameras able to collect many frames per second over extended periods of time. Although thermal imaging is a widely used technique in avian research 25, size and expense of thermal imaging systems have restricted its use in the wild. In this study the system was highly portable, cost approx. &#163;6,000 and provided high resolution thermal video capable of non-invasively capturing accurate temperature data from free-living animals without the need of handling birds. Recording at multiple frames per second allows measurements of high resolution time series of peripheral temperature and thus provides the possibility to explore the dynamic of the acute stress response. This is difficult to achieve with the conventional glucocorticoid sample that is constrained by the number of samples that can be taken within a period of time. Here we have extracted a time series of eye-region temperature with an interval of 1 sec, which was sufficient to demonstrate the eye-region temperature response to an acute stressor (capture), but higher temporal resolution would also be possible. The accumulation of a large number of images, however, requires some level of automation in the extraction of information from the images. The protocol describes a simple semi-automated process to extract the maximum temperature from each image. We were able to do this as the region of interest, the eye-region, was always the warmest spot in the image. Analysis could be more problematic if regions of interest are more complex and may require custom designed pattern recognition software (e.g.,34). In this study some manual filtering was required for situations where the eye-region temperature was over- or under-estimated but these were easily detected, but of course further automation is desirable. The measurements of stress-induced peripheral cooling by thermal imaging provides a valuable addition to other physiological measures of stress and the non-invasive aspect of this technique is highly advantageous for further studies involving captive and wild animals.
Capturing the complete stress response, infrared thermography clearly has great potential as a tool for stress assessment. To become a non-invasive alternative to established hormonal and core temperature assays though, it will be necessary to cross-validate amongst these measures and determine if skin temperature shows the same proportionality with stressor intensity, i.e., the extent of the skin temperature response can reflect the strength of the stressor. Future research should also address whether skin temperature captures chronic stress. Whilst SIH in response to an acute stressor is expected to be transient, recurrent exposure to physical or psychological acute stressors can generate chronic elevation in core body temperature 35,36. Whether ongoing vasoconstriction contributes to this core elevation has not been tested explicitly. However, correlative studies on humans suffering from chronic stress-related illness do show reduced finger temperatures 37. A final attribute to explore is valence: the ability to distinguish positive from negative events. Hormonal assays cannot distinguish valence 5, with glucocorticoid levels appearing to reflect level of excitation rather than stress specifically. Research on comb temperature in domestic chickens exposed to aversive and positive stimuli suggest skin temperature may be similarly akin to general arousal 22,29. However, in humans, different emotional states elicit regionally specific changes in skin temperature, for example periorbital warming and cheek cooling when startled 38, but overall decreased skin temperature when laughing 39. Comparisons between different regions may yet reveal emotional state. Using the recommendations laid out in this paper, it will be possible to address all of these questions, and validate skin temperature as a non-invasive marker of stress.

Stress is a central concept in biology, describing the emergency response of an organism in response to an environmental challenge attempting to restoring homeostasis 1,2. Under stress, blood glucose, fatty acids and amino acids levels, heart rate, respiratory rate and metabolic rate all increase, and blood is diverted from the periphery to the core organs 2. This generic pattern of physiological changes primes the animal to be able to respond quickly and adaptively to an array of social and physiological challenges. Whilst recognizing and understanding stress is at the heart of both pure and applied animal research, assessing stress in unrestrained animals remains a challenge.
A widely used physiological marker of stress is an increase in the levels of circulating glucocorticoid hormones, such as cortisol and corticosterone 1,2. A great strength of this approach is that their concentrations increase in proportion to stressor intensity, allowing stress to be quantified. However, glucocorticoids are not &#8216;stress&#8217; hormones per se, but mobilisers of energy stores 2. As such glucocorticoid levels change also with energy requirements, time of day, age and reproductive status 3,4, as well as in response to apparently positive situations, such as mating opportunity 5. Glucocorticoid levels, therefore, must be interpreted cautiously and within a context. Measuring the glucocorticoid response to acute stress has also some limitations. An acute stressor triggers a dynamic response of initially increasing glucocorticoid levels and subsequently returning to a baseline level 1,2. Glucocorticoid samples are typically obtained invasively, by blood sampling which is a stressor in itself and therefore can affect the measured glucocorticoid levels 6. Moreover glucocorticoid levels can only be measured at one or very few time points, which may not capture variation in peak levels and timing or response duration, limiting our ability to examine change through time of the dynamic stress response within individuals. Non-invasive methods of hormone sampling, such as from feces 7, hair or feathers 8, measure average glucocorticoid levels over a longer time scale, of days or months, although useful to study chronic stressors, are not applicable to the study of acute stress. As even the best established methods offer only a limited perspective on variation in acute stress amongst individuals there is a need for an alternative methodology in physiological stress measurements.
As the stress response involves a number of physiological effects there are other candidates that could indicate stress. Among others, sympathetically-mediated vasoconstriction channels blood from the periphery to the body core. This concentration of blood and hence heat, along with various forms of stress-induced thermogenesis, warms the core 9. As such, core warming, termed stress-induced hyperthermia (SIH) has also been used as a marker of acute stress in pharmaceutical research 10. SIH typically raises core body temperature by 0.5&#8211;1.5 &#176;C within 10&#8211;15 min of an acute stressor 10. It is a relatively well documented phenomenon that a commonly applied experimental stressor of catching and handling an animal can raise core body temperature in a range of mammal and bird species 11&#8211;17. Importantly, SIH correlates with other established indicators of stress, such as heart rate 18, glucocorticoid levels 19 and behavior 20,21. And like glucocorticoid levels, core temperature has been linearly related to stress level in some species 22. However, as with blood sampling, the measurement of core temperature is invasive in itself, requiring the insertion or implantation of a probe 10. Baseline core temperature gradually increases each time when the stress-inducing handling or probe insertion must be repeated 23. Recent developments in temperature recording equipment that allow accessing data remotely may provide a solution, at least for large animals 24.
Within the same mechanism that generates SIH, though, lies yet another potential marker of stress: the peripheral vasoconstriction that moves blood, and with it heat, to the core simultaneously cools the skin 17. Unlike core temperature, skin temperature can be measured completely non-invasively, by infrared thermography (IRT). IRT converts the infrared radiation emitted from the surface of an object into temperature 25. As an animal&#8217;s surface typically has few bare patches from which skin temperature can be derived directly, it requires access to naturally exposed skin (e.g., area around the eye) or creating a bare patch by trimming fur or feathers. Provided access to a suitable exposed area of skin and the animal can be kept in the field of view of the camera, IRT cameras may be used to collect continuous skin temperature measurements remotely, possibly allowing the complete temperature response to be filmed and compared between individuals. While it has already been shown in chickens that surface temperature responds to acute stress 26, the novelty of this study is that it measures surface body temperature of wild animals in a finer temporal resolution than previous studies and also shows that the expected skin temperature drop can be detected in the field where temperature, humidity and weather are variable. The aim of this paper is to describe the necessary methodology to measure skin temperature of an unconstrained animal using thermal imaging. We use capture to induce a mild acute stress in free-living blue tits (Cyanistes caeruleus, Linnaeus 1758) and describe how to capture suitable imagery, analyze the images and process the data to produce temperature response curves against time that demonstrate a significant cooling of the birds&#8217; skin temperature in response to the mild stressor. This can provide a valuable alternative method to assess stress in wild and captive animals useful to a wide range of researchers from ecologists, conservation biologists and physiologists to animal welfare researchers.
The study was carried out in the period between 17th December 2013, and 4th January 2014 in a deciduous oak (Quercus spp.) forest at the Scottish Centre for Ecology and the Natural Environment (SCENE), University of Glasgow at the eastern shore of Loch Lomond, west central Scotland (56.13&#176;N,&#160;4.13&#176;W). The protocol involves three main steps: (1) Setting up suitable conditions under which to capture thermal images from a free-ranging animal, (2) applying an acute stressor while taking a thermal video, and (3) extracting and processing data from the thermal images that then can be used to characterize the animal&#8217;s response to the acute stressor. In our case we attracted a free-living passerine, the blue tit to a feeding box (filming set-up) where conditions for thermal image capture were optimized, and then applied a capture stress by closing the feeding box remotely when the bird was inside. The description of thermal image capture and data extraction is specific to the equipment we used, and may vary between thermal imaging systems. Data processing is described using an open-source software.

<table><tbody><tr><td>Thermal Imaging Camera</td><td>FLIR</td><td>A65 (f=25 mm)</td><td>Compact (106 x 40 x 43 mm) and low cost</td></tr><tr><td>Thermal Image Analysis Software</td><td>FLIR</td><td>ResearchIR v3.4</td><td>Allows high speed thermal video recording and thermal pattern analysis</td></tr><tr><td>Temperature logger</td><td>Gemini Data Loggers</td><td>Tinytag Talk 2 TK-4023-PK</td><td>Monitors from -40 to +125 &amp;#176;C using accompanying temperature probe</td></tr></tbody></table>

thermal imaging to study stress non-invasively in unrestrained birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

Results from 9 free-living blue tits are presented that illustrate the information that can be obtained from thermal imaging and demonstrate that the predicted signal of stress in the bird&#8217;s eye-region temperature can be detected in free-ranging animals. Each bird was filmed for an average of 5.1&#177; 0.9 sec (n=9) before the box&#8217; door was closed. This allowed the calculation of the undisturbed bird&#8217;s eye-region temperature (baseline temperature) to which all subsequent measures can be referred to. In tests conducted on 20 sec thermal video clips of undisturbed blue tits arriving and feeding within a trap (n=9 birds different from the ones used for the application of a mild stressor), correlations of r &#62; 0.7 were found to exist between the maximum eye-region temperature recorded during the first 5 sec, and the maximum recorded in the first 10, 15 and 20 sec (Figure 3). This was interpreted as indicating that the maximum value recorded in the initial few seconds after trap entry was representative of the longer-term level, and the mean eye-region temperature during approximately 5 sec immediately preceding box closure were used as the baseline value. The blue tits in this sample had a mean baseline eye-region temperature of 38.4 &#177; 0.5 &#176;C (mean &#177; SE) and ranged from 35.8 to 39.9 &#176;C (n=9). Once the bird fed for 5 sec to allow the calculation of the baseline eye-region temperature, the box door was closed by the experimenter. All birds noticed the closure of the box as evidenced by their attempted escape flights.
Upon closure of the trap, the eye-region temperature dropped rapidly and reached a minimum eye-region temperature ~1.3 &#176;C below the baseline temperature after ~10 sec (Figure 4). Figure 4 shows the composite curve based on the maximum eye-region temperature of all nine individuals average for every second. As the timing and the magnitude of the eye-region temperature drop varies between individuals the composite curve masks the true extent of the temperature response. The mean temperature drop calculated for each individual separately is 2.0 &#177; 0.2 &#176;C and the lowest point is reached after 9.4 &#177; 2.8 sec (mean &#177; SE). From then on, eye-region temperature gradually returned towards baseline eye-region temperature over the following 2-3 min but did not quite reach the baseline by the end of the trial.
<img alt="Figure 1" src="/files/ftp_upload/53184/53184fig1.jpg" /> 
Figure 1. The filming arena. The set-up in the field was designed to attract the birds to the field of view of the camera where a mild acute stressor could be applied, using a feeding box. The set-up consisted of a 25 by 14 cm and 16.5 cm high box built from plywood, with a front panel of galvanized wire mesh with an aperture size of 1 by 2.5 cm. The mesh allows infrared radiation to pass through, making it possible to film birds whilst inside the set-up. Wire mesh was chosen, as glass and most plastics do not transmit infrared radiation. A hole of 60 mm diameter was cut in the left end wall to allow bird entering the set-up and gain access to the food placed at the right end of the box. A temperature logger probe sandwiched between two squares of black insulation tape (marked by black arrow) is attached to the top right corner of the front panel to record a reference temperature. The mild acute stressor is applied to the bird inside by closing the box. Pulling on a fishing line attached to a rotating door at the entrance hole allowed the experimenter to close the box when a bird is inside. The thermal imaging camera set in front of the trap records the whole sequence of events. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53184/53184fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53184/53184fig2.jpg" /> 
Figure 2. Thermal Image of a Blue Tit. The majority of a passerine&#8217;s body is well insulated by feathers (or to a lesser extent by leg scales), but the skin around the eye is exposed and radiates most heat under normal circumstances, and is surrounded by cooler integument or background. This is shown in this thermal image by the yellow color (highest temperature) around the eye, the orange, red, purple and blue color signify cooler and cooler temperatures. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53184/53184fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53184/53184fig3.jpg" /> 
Figure 3. Repeatabilities of baseline temperature measured over different periods of time. Correlation coefficients between the maximum temperature recorded in the first 1-8 sec of a clip and the &#8216;mean of the maximums&#8217; within 0.5&#186; of the highest value recorded for the first 10, 15 and 20 sec of the clip (represented by red, blue and green lines, respectively). All clips were 20 sec in duration (n=9). During initial analysis using a larger dataset, a number of the &#8216;means of the maximums&#8217; were calculated from just 1 value, and gave the same value for rho across all comparisons. These clips were removed from the analysis, leaving only those where means were calculated from more than 3 measurements. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53184/53184fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53184/53184fig4.jpg" /> 
Figure 4. Response of the eye-region temperature to trapping. There is a very clear and pronounced signal of the trapping event reflected in the change of eye-region temperature over time. To compare across individuals that vary in their baseline temperature, residual eye-region temperature, as the difference of the actual temperature and that individual&#8217;s baseline temperature, is plotted on the vertical axis. This is plotted against time with the closure of the trap set as 0 and the bird removed from the trap after 3 min. The red line shows the average residual temperature and the grey band represents the 95% confidence interval. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53184/53184fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds at JoVE.com

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

thermal-imaging-to-study-stress-non-invasively-in-unrestrained-birds

Ateneo de Manila University

Research

JoVE Journal

Neuroscience

13.4K Views.  University of Glasgow. There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Video: Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

Medicine

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

Behavior

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Impedance Pneumography for Minimally Invasive Measurement of Heart Rate in Late Stage Invertebrates

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by environmental temperature, this has the potential to alter thermal physiological performance in a variety of marine organisms. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography to understand how cardiac performance in late stage invertebrates changes under acute thermal stress. The protocol presents a minimally invasive technique that allows for real-time collection of heart rate during a temperature ramping experiment. Data are easily manipulated to generate an Arrhenius plot that is used to calculate Arrhenius break temperature (ABT), the temperature at which heart rate begins to decline with increasing temperatures. This technique can be used in a variety of late stage invertebrates (i.e., crabs, mussels, or shrimps). Although the protocol focuses solely on the impact of temperature on cardiac performance, it can be modified to understand the potential for additional stressors (e.g., hypoxia or hypercapnia) to interact with temperature to influence physiological performance. Thus, the method has potential for wide-ranging applications to further understand how marine invertebrates respond to acute changes in the environment.

Measuring heart rate during a thermal challenge provides insight into physiological responses of organisms as a consequence of acute environmental change. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography as a relatively noninvasive and nonlethal approach to measure heart rate in late stage invertebrates.

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by ...

Environment

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Bioengineering

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...